Prescription drugs and cosmetics are used for extensive periods of time, and their development requires demonstration of long-term safety. Major causes of damage include hepatotoxicity, cardiotoxicity, and neurotoxicity, and nephrotoxicity. Current methods to detect toxicity rely on large number of cells, and dozens of end-point fluorescent, colorimetric, or histological assays dramatically increasing the cost of toxicity screening. Inherently, these techniques provide limited toxicokinetics information. While this is a marginal concern for evaluation of acute toxicity, toxicokinetic information is critical for safety evaluation of prescription drugs and cosmetics. Such a repeated-dose response assay requires two months of daily administration to demonstrate chemical safety of chronic exposure for up to one year. Until now, only animal models could serve these requirements, as it is difficult to maintain cells in culture for over 28 days. However, animal model are inaccurate with 70% of the compounds found toxic in animals are not toxic in humans, and vice versa.
Microfluidic liver-on-chip devices offer an alternative to animal experiments as they can mimic the native microenvironment and support long-term function under continuous perfusion. One critical advantage of microfluidics is the ability to expose cells to a stable stimulation over time, eliminating the rapid loss of signal due to non-specific adsorption and metabolism that characterizes both static in vitro assays and in vivo. Stable microfluidic stimulation permits the acquisition of reliable information about the effect of a specific dose, rather than the response of cells to a rapidly changing drug concentration. Regrettably, current devices still rely on end-point histological or molecular analysis of function to assess the toxicological effect of a molecule of interest, e.g., a drug. It is clear that real-time measurement of cell viability is needed.
Oxygen uptake is a critical measurement of mitochondrial function and metabolic activity (Green and Reed 1998; Han et al. 2013). There is a need for reliably measuring oxygen on the microscale. Regretfully, measurements of fluorescence intensity, such as by particles whose fluorescence is quenched in the presence of oxygen, are affected by small changes in focus, particle migration, and cell movement making these types of probes unreliable for real time measurements.
One of the main intracellular targets of drug-induced liver injury is mitochondrial function, either through direct damage to the respiratory complex (e.g. NAPQI) or though secondary mechanisms such as ER stress (e.g. tunicamycin). Currently, end-point assays such as MTT or JC1 staining are used to evaluate mitochondrial function or its membrane potential, respectively. An alternative approach will monitor oxygen consumption directly using classical Clark-type electrodes or oxygen-quenchable fluorophores (e.g. ruthenium compounds) (Papkovsky and Dmitriev 2013; Ramamoorthy et al. 2003). However, Clark-type electrodes do not meet the needs of miniature in vitro assays as the electrochemical reaction consumes oxygen during measurement and needs frequent recalibration. On the other hand, optical oxygen sensors are more reliable but have to be physically inserted into the sample or coated on the bottom of the culture chamber. Regretfully, fluorescence intensity measurements are affected by small changes in focus occurring due to movement of the mechanical stage and cells, limiting the utility of optical oxygen sensing (Vanderkooi et al. 1987).
Therefore, there is a need to develop a system for the continuous monitoring of cellular toxicity (e.g., for up to two months) in vitro using human cells. Such a system could mimic human physiology, providing a perfused, three-dimensional microenvironment, in which cellular function is maintained at high levels.